Project Summary/Abstract Adult cardiomyocytes (CMs) are specialized to forcefully contract and relax billions of times during an animal's lifetime. A number of adaptations allow adult CMs to fulfill this role, including: large, elongated shape; highly organized network of cell-cell junctions; reliance on oxidative metabolism; nearly crystalline array of sarcomeres; characteristic organization of calcium release units around a network of plasma membrane invaginations known as T-tubules; cell cycle exit; and expression of adult CM-specific contractile, calcium handling, and ion channel genes. In mouse, most of these adaptations are acquired in the first three postnatal weeks. Little is known about the molecular regulation of the CM maturation program. This information gap limits development of rational approaches to stimulate maturation of stem-cell derived CMs. Furthermore, it is likely that congenital heart disease mutations or abnormal cardiac stress caused by congenital heart malformations impact CM maturation, with implications for long term myocardial performance of congenital heart disease patients. Our long term goal is to understand the regulatory program that governs CM maturation. Our preliminary data show that GATA4 and GATA6 (GATA4/6) transcription factors are essential for CM maturation. In addition, a forward genetic screen uncovered TAF3, a component of the RNA Polymerase II pre-initiation complex and a reader of H3K4me3 epigenetic marks, as a novel maturation regulators. Using a number of unique and novel tools and approaches, this proposal will investigate mechanisms that control CM maturation. Aim 1 will dissect the mechanisms by which GATA4 and GATA6 regulate enhancer activity during CM maturation. Aim 2 will further characterize TAF3 mutants and dissect the mechanisms by which it regulates maturation. Aim 3 will apply our innovative in vivo forward genetic screen to discover signaling molecules and congenital heart disease genes that are essential for CM maturation. The coordinated transformation of fetal CMs to their mature counterparts is among the least well understood aspects of cardiac development. Success with this proposal's aims will advance our knowledge in this unexplored frontier of cardiac development.